The invention relates to melanins, suitable to be used in food industry, pharmacy, medicine and bioelectronics.
The invention relates also to a process for production of a narrow molecular fraction of melanin from raw materials of vegetable origin, so called phytomelanin, which exhibits defined and reproducible physico-chemical properties and higher biological activity than that of known, described vegetable melanins, and which is suitable to be practically used in industry and pharmacology.
Melaninsxe2x80x94a general name for groups of high-molecular black and brown pigments, arising in the course of oxidation and polymerisation of phenols. Melanins occur normally in nature, and they are one of the most often occurring zoochromes. They occur in hair, eyes, skin, inner organs and so they are essentially mostly localised in the surface parts of organisms. Coloration of dark seeds, berries, flower leaves, and plants, men suntan, skin of blacks, many kinds of animals is mostly due melanins, as mentioned, for example, in Nicolaus R. A.: Melanins, Hermann, Paris 1968, p. 310; Lyiach S. P., Ruban R. D.: Mikrobnyie melaniny, Nauka, Moscow 1972, p. 184; and Bidzilja N. I.: Svobodnyie radikaly v oblutschennykh rastenyiach i semenakh, Naukova dumka, Kiev 1972, p. 210. The term xe2x80x9cmelaninxe2x80x9d itself originates from a Greek word, and it means xe2x80x9cblackxe2x80x9d. Melanins are unique biopolymers which exhibit in a living organism protection function against UV radiation, ionising radiation, high and low temperatures. Melaninogenesis has been at present often presented as a complex adaptation of living organisms at the border of adaptability of life. It is possible to find unique examples of resistance of living organisms to geophysical and geochemical factors in extreme situations. These include first of all upland regions, where blackpigmented fungi constitute the only microflora at 4 to 5 km height, and also hot sandy and cold stony deserts of several regions [Lyiach S. P.: Mikrobnyi melaninogenez i yiego funktsii, Nauka, Moscow 1981, p. 274.; Ostrovskayia M., Dontsov A.: Fyziologitcheskyie funktsii melanina v organizme, Fyziologyia tscheloveka 1985, p. 670-679]. There are also organisms known which are stable when irradiated with sublethal doses of the order of 900 Krad. Inertness to xcex3-radiation decreases with the pigment loss, too. The question of melanin function in paleobiologicali aspect is extremely interesting. Highly melanised fungi spores occur in great amounts unusually often in the layers of the beginning of at xe2x80x9cCretaceous periodxe2x80x9d when many species of animals and plants died out. This period is identical with the period of the Earth""s crossing the xe2x80x9cmagnetic zeroxe2x80x9d, and thus of its inability to protect itself against cosmic radiation [Bidzilja N. I.: Svobodnyie radikaly v oblutschennykh rastenyiach i semenakh, Naukova dumka, Kiev 1972, p.210; Lyiach S. P.: Mikrobnyi melaninogenez i yiego funktsii, Nauka, Moscow 1981, p. 274; Ostrovskayia M., Dontsov A.: Fyziologitcheskyie funktsii melanina v organizme, Fyziologiya tscheloveka 1985, p. 670-679]. Hence, also a scientifically proven basis exists that melanins were the xe2x80x9cbeneficialxe2x80x9d material which helped chemical evolution of some of polymeric prebiological structures. The above given possibility follows from the character of the process of synthesis of these substances, and from the properties of contemporary melanins. Great attention should be paid to the easiness with which are the pigments synthesized, when conditions are modelled which are supposed to have existed on the Earth in the period of origin of complicated substances from aromatic structures [Blois M. S.: Proischozhdenyie predbiologitscheskikh sistem, Mir, Moscow 1966, p. 494; Pavlovskayia T. E.: Abiogenez i natschalnyie stadii evolutsii zhizni, Nauka, Moscow 1968, p. 216; Blois M. S.: The melanins, their synthesis and structure, Photochem. and Photobiol. Rev. 3, 151, 1978: Swan G. A.: Current knowledge of melanin structure, Pigment cell, Vol.1, Harger, Basel 1973, p. 151].
Classification of Melanins
Depending on the biological subjects which synthesise them, melanins are divided in three basic groups: microbial, animal and vegetable. There exist also synthetic melanins which arise by autooxidation of 3,4-dihydroxydiphenylalanine (DOPA-melanin), as indicated, for example, by Mason H. S. in Pigment Cell Growth, Acad. Press, NY 1953, p. 235; Peers E.: Hystochemistry, IL, Moscow 1962, p. 640; Keretz D., Ann. intab. dermatol. din. esperimentele 1961, p. 268; and Thomas M.: Modern methods of plant analysis, Springer Verlag 1953, 4, p. 661. Microbial melanins are met only at some microorganisms, especially those belonging to the genera: Bacillus, Pseudomonas and Azatobaster (Azotobacter). These are black and brown, sometimes red-brown pigments which are, in general, insoluble in organic solvents, soluble in bases with non-specific spectral characteristics. Many facts prove that oxidation processes are the base for the origin of bacterial melanins. Attention should be paid to the fact that an absolute majority of microorganisms which synthesise the pigments, belong to aerobic forms. The animal melanins are localised in surface tissuesxe2x80x94skin, hair, animal hair, feathers and retina. The vegetable melanins have been described only rarely. It is known that they occur in surface tissues of some seeds and fruits. Up to now three methods of vegetable melanins isolation are known and described, namely of the phytomelanin from Vitis Vinifera L. Nevertheless, the preparations are summary products showing broad spectrum of physico-chemical properties and, consequently, the product cannot be used as a drug base, as indicated in Zherebin J. L. et al.: Sposob polutschenyia vodorostvorimogo melanina, t. A. S. SSSR patent Nr. 939446, 1983; Sendega R. V., Venger L. A., Baklanova L. V.: Sposob polutschenyia enomelanina, patent A. S. SSSR Nr. 1345606, 1987; and Godzenko A. I. et al.: Sposob polutschenyia enomelanina, patent RU 07 09 93, bl. Nr. 33-36. The best described and known among the melanins is the so called synthetic melanin or DOPA-melanin which arises by autooxidation of 3,4-dihydroxydiphenylalanine (DOPA). The DOPA oxidation is going on in such a way and through such stages like the fermentative autooxidation of tyrosine in living organisms which results in the rise of animal and microbial melanins. A scheme of the process is given in Villee Claude A., Dethier Vincent G.: Biological principles and processes, Philadelphia-London-Toronto 1971, p. 822; and Brechtlovxc3xa1, Hal{haeck over (c)}xc3xa1, Chandoga et al.: Lekxc3xa1rska biochxc3xa9mia I. (Medicinal biochemistry I), Asklepios 1992, p. 228. 
Chemical Structure and Melaninogenesis
Chemical structure of natural melanins has not been established yet, because they have very complicated polymeric structure and type diversity. Therefore, we could not obtain exhaustive description even for those pigments which have been investigated for several years. At present no unanimous opinion exists on the problem which compounds correspond to the term xe2x80x9cmelaninxe2x80x9d. Mason describes melanins as high-molecular polymers which arise in the course of enzymatic oxidation of phenols, especially of pyrocatechol, 3,4-dihydroxyphenylalanine, (DOPA) and 5,6-dihydroxyindole [Mason H. S. in Pigment Cell Growth, Acad. Press Inc., NY 1953, p. 235]. 
A related essay has been written by Nicolaus: xe2x80x9cNatural melanins are complicated macromolecules which arise in the course of enzymatic oxidation of ortho-diphenols, mostly unsubstituted, such as 5,6-dihydroxyindole, pyrocatechinole and 1,8-dihydroxynaphthalenexe2x80x9d [Nicolaus R. A.: Melanins, Hermann, Paris 1968, p. 310]. 
Peers and Keretz describe melanin pigments arising in the course of oxidation of aromatic amino acids: tyrosine and dihydroxyphenylalanine [Peers E.: Hystochemistry, IL, Moscow 1962, p. 640; Keretz D., Ann. intab. dermatol. din. esperimentele 1961, p. 268]. Thomas proposes to consider as melanins only nitrogen containing pigments, i.e. derivatives of 5,6-dihydroxyindole which occurs either in an oxidised or in a reduced state [Thomas M.: Modern methods of plant analysis, Springer Verlag 1953, 4, p. 661].
At present two basic theories on the origin and structure of zumelanins exist. The first states that zumelanins are essentially homopolymers of indole-5,6-quinone [Pulman B., Pulman A.: Kvantovayia biokhimyia, Mir, Moscow 1965, p. 654]. The second, Nicolaus""s interpretation, results from many experiments performed assuming that in the mechanism of melanogenesis free radical polymerisation of various monomers takes place. The polyfunctionality of monomers, the absence of precise bond structure between the radicals lead to the synthesis of polymers having nonuniform content and organisation structure. This finally lead to the opinion that in nature there probably do not exist any two absolutely identical melanin pigments [Nicolaus R. A.: Melanins, Hermann, Paris 1968, p. 310]. Therefore, melanin is a three-dimensional polymer for which the number of possible structures is equal to the number of types of melanin molecules in nature. For many years the pigment of the Ustilago maydis fungus spores has been considered as the basic model. It is practically the only (microbial) melanin the structure of which has been, in general, examined and confirmed (Formula IIa). 
Nicolaus concludes that complicated cyclic structures with condensed rings, occurring in different oxidation states, arise in the course of polymerisation of pyrocatechinole to melanin [Nicolaus R. A.: Melanins, Hermann, Paris 1968, p.310; Lyiach S. P., Ruban R. D.: Mikrobnyie melaniny, Nauka, Moscow 1972, p.184].
The second, essentially examined melanin is the black pigment arising in ripe spores of the Aspergillus niger fungus. It belongs to the group of so called alomelanins, and it is called Aspergillus niger-melanin [Nicolaus R. A.: Melanins, Hermann, Paris 1968, p.310; Blois M. S.: Proischozhdenyie predbiologitscheskich sistem, Mir, Moscow 1966, p.464]. 
At present it is possible to assume that the base for melaninogenesis in living organisms consists in a process which is based on fermentative oxidation of tyrosine via DOPA in the presence of tyrosinase to dopaquinone of which pigment arises through a number of oxidations, decarboxylations and conjugations. This is the xe2x80x9cclassical wayxe2x80x9d.
Anyway, the synthesis sometimes passes a different path and the melanogen may be not only tyrosine, but also other phenols, for example pyrocatechinole. In spite of that it should be remembered that the genetically conditioned ability to form tyrosinase and melanin pigments in living organisms is highly stable on the whole [Brechtlovxc3xa1, Hal{haeck over (c)}ik, Chandoga et al.: Lekxc3xa1rska biochxc3xa9mia I (Medicinal biochemistry 1), Asklepios 1992, p. 228; {haeck over (S)}kxc3xa1rka B., Feren{haeck over (c)}ik M.: Biochxc3xa9mia, Alfa, Bratislava 1992, p. 846].
Identification of Melanins
One of the reasons for insufficient examination of melanins is the problem of their isolation from biological objects, because these pigments are insoluble in most of known organic and mineral solvents [Nicolaus R. A.: Melanins, Hermann, Paris 1968, p. 310; Lyiach S. P.: Mikrobnyi melaninogenez i yiego funktsii, Nauka, Moscow 1981, p. 274; Blois M. S.: The melanins, their synthesis and structure, Photochem. and Photobiol. Rev. 3, 151, 1978]. Moreover, melanin is present in biological objects in vivo, and it readily binds to other polymers: proteins, polysaccharides, lipids, other pigments and admixtures.
Difficulties with and problems of isolation of melanins consist also in their colloid character and inability to crystallise. As a consequence two basic methods of melanin pigments isolation exist.
The first group of methods consists in melanin extraction with suitable solvents, and then in elimination of accompanying admixtures. The only, nearly universal solvents for melanins are 0.5 to 1.0 M aqueous solutions of the bases NaOH and KOH. Such properties of melanins are conditioned by their polyphenolic structure. Therefore, also alkaline extraction is used for isolation of melanins from cells and tissues of animals, microbial and some vegetable subjects.
The second group of melanin extraction methods is presented as a method in which all other materials except melanin are eliminated. This elimination of useless materials is performed by hydrolysis with an acid and washing with suitable solvent until the required preparationxe2x80x94melaninxe2x80x94remains [Lyiach S. P., Ruban R. D.: Mikrobnyie melaniny, Nauka, Moscow 1972, p. 184].
As a consequence, no standard method, suitable for all kinds of natural melanins, can be proposed and described, because chemical complexity and diversity of biological materials and specialities of melanins alone require individual approach. For example, for the extraction of ustilagomelanin another Process for preparative technique is used. Spores of the Ustilago maydis fungus are treated by concentrated HCl and then, using various solvents, admixtures are removed. Melanins of some subjects are essentially easily isolated by xe2x80x9csoft methodsxe2x80x9d. This concerns, for example, melanin of bull""s eye iris or melanin of the ink of the octopus Sepia officinalisxe2x80x94sepiomelanin. The preparations are isolated without treating with an acid or a base, because they occur in the biological subject in the form of sodium salts. But there are only very few such examples. In principle, the method of alkaline extraction with the following precipitation by an acid is used for melanins of animal, microbial and vegetable origin [Nicolaus R. A.: Melanins, Hermann, Paris 1968, p.310].
The melanin content in the cells of fungi is varying in a broad range: from 2 to 3% by weight up to 35 to 40%. The melanin content in vegetable subjects is considerably lower, namely from 0.2 to 0.3% by weight up to 6 to 8% by weight.
One of the identification tests for melanins are the VIS spectra. Melanins exhibit bright spectral absorption lines, one in the wavelength region of 1600 to 1700 cmxe2x88x921, the second in the region of 3000 to 3500 cmxe2x88x921. The spectral lines correspond to carbonyl groups (absorption in the region of 1700 cmxe2x88x921), carbon-carbon bonds (absorption in the region of 1600 cmxe2x88x921), NH4 and OH-groups (absorption in the region of 3300 to 3500 cmxe2x88x921).
Melanins of natural, as well as of synthetic origin show a characteristic UV-spectrum in which they show neither any peaks nor clear absorption bands, especially in the range of short wavelengths. A similar characteristics is in the region of visible part of the spectra with an inclined diagonal within the limits of xe2x88x920.0019 to xe2x88x920.0040. Such optical characteristics are typical for melanins.
Also various chemical reactions are characteristic, by which it is possible to judge whether the melanin monomers occur in the preparation:
a/ insolubility in water and in most organic solvents,
b/ full solubility in 0.5 M NaOH or KOH,
c/ precipitation from solutions in the presence of FeCl3,
d/ discoloration if strong oxidation agents (KMnO4, H2O2) are used,
e/ ability to regenerate ammonia solution of AgNO3.
The most important characteristics of melanin pigments is the presence of paramagnetic centres with a concentration of 1015 to 1018 spin/g [Nicolaus R. A.: Melanins, Hermann, Paris 1968, p. 310; Bidzilja N. I.: Svobodnyie radikaly v oblutschennykh rastenyiach i semenakh, Naukova dumka, Kiev 1972, p. 210; Ostrovskayia M., Dontsov A.: Fysiologitcheskyie funktsii melanina v organizme, Fyziologyia tscheloveka 1985, p. 670-679].
From the biological point of view determination of the melanin nature of pigments consists in the isolation of tyrosinase and of its substrates in the subjects given, and also determination of direct connection between tyrosinase activity and melaninogenesis (pigmentogenesis).
Therefore, identification of melanin based on one or two tests is not unequivocal, so that a whole complex of tests must be used. The presence of an unoccupied bottom energy level in melanins and their ability to xe2x80x9ctrapxe2x80x9d unpaired electrons from the environment leads to the fact that melanin exhibits radioprotective properties absorbing unpaired electrons of free radicals, arising in the systems under the action of ionising radiation [Bidzilja N. I.: Svobodnyie radikaly v oblutschennykh rastenyiach i semenakh, Naukova dumka, Kiev 1972, p. 210; Godzenko A. I. et al.: Sposob polutschenyia enomelanina, patent RU 07 09 93, bl. Nr. 33-36].
In the last years results of experimental studies have been published which have confirmed the radioprotective properties of melanins. Melenins exhibit the properties when naturally occurring in the subject, but also when they are artificially introduced into a living organism [Hill H. Z., Hill G. J.: Eumelanin Causes DNA Strand Breaks and Kills Cells, Pigments Cell Research 1, 163-170, 1987; Hill H. Z., Peak J. G., Peak M. J.: Induction of DNA-protein crosslinks in melanotic cloudman S91 mouse melanoma cells by monochromatic 254 and 405 nm light, Pigment Cell Research 2, 427-430, 1989; Tsuneaki Chida, Hugh D. Sisler: Effect of inhibitors of melanin biosynthesis on appresorial penetration and reductive reactions in Pyricularia oryzae and Pyricularia grisea, Pesticide Biochemistry and Physiology 29, 244-251, 1987; Jacobsohn M. K., Dobre V. C., Branam Ch., Jacobsohn G. M.: Oxidation of 2-hydroxyestradiol and its incorporation into melanin by mushroom tyrosinase, J. Steroid Biochem. 31(4A), 377-385, 1988; Giovanni Sichel: Biosynthesis and function of melanins in hepatic pigmentary system, Pigment Cell Research 1, 250-258, 1988]. The experiments which have been performed on black cells containing melanin of the yeast Nadsomiela nigra have shown that these are considerably more resistant to radiation than the cells of yeasts which contain no melanins. By artificial cultivating of yeasts in biological medium, containing hexachloroacetone which is an inhibitor of melaninogenesis, a culture has been cultivated which has completely lost resistance against radiation [Chrulyiova I. M.: lssledovanyie struktury i svoystv melanina i yego syntetitscheskikh analogov, Ref. Zh., Moscow 1973, p. 20; Baraboyi V. A.: Biologitscheskoyie deystvyie rastitelnykh fenolnykh soyiedinenyi, Naukova dumka, Kiev 1976, p. 260; Zherebin J. L. et al.: Farmakologitscheskyie svoystva enomelaninovykh pigmentov, Doklady AN SSSR, seryia B 1984, pp. 64-68].
The results of practical experiments and review of the literature available make it possible to develop a melanin preparation which would serve as an effective radioprotector for living cells of organisms, produced on the basis of natural materials and products of metabolism.
Antitumour Activity of Melanin Pigments
At present most of research workers and scientists believe that a cancer cell differs from a normal cell not by the fact that it lacks some specific substances, but by the ratio of components of biochemical systems, belonging to a normal cell. The works of N. M. Emanuel et al. have confirmed that a change of concentration of free radicals in biochemical components of the cell is essential for negative growth of the cell and, therefore, antioxidants must influence the progress of the processes. Based on this, the authors could assume that such physico-chemical property of melanins like antioxidation activity is an important indicator of the process of the cell metabolism. It is a basis for the ability of phenol groups to react with free radicalsxe2x80x94active centres of the cell biochemical system. An elementary act of co-operation of an inhibitor with a free radical R leads in the system to creation of an inhibitor radical which is more stable and less reactive than the radical R [Chrulyiova I. M.: Issledovanyie struktury i svoystv melanina i yego syntetitscheskikh analogov, Ref., Moscow 1973, p. 20].
This hypothesis has been confirmed in scientific studies. At that time synthetic, natural (animal) and biosynthetic melanins were chosen as a subject for the investigation. The animal melanin was obtained from the mice melanoma Harding-Passa by an acidic-alkaline method. The biosynthetic melanin was synthesized from DOPA in the presence of tyrosinase, isolated from the mice melanoma Harding-Passa [Chrulyiova I. M., Berlin A. A.: Protivoopukholyievayia aktivnost syntetitscheskikh, biosyntetitscheskikh i prirodnykh melaninov, Izvestiya AN SSSR 1973, Nr. 3, pp. 438-442]. Based on the performed experiments it could be proven that melanins are not carcinogenic, and the results allowed to confirm that melanins have the ability to show antitumour activity with doses of 150 to 250 mg/kg, reaching the effect of 50 to 60% retardation of the tumour growth.
Immunogenic Activity of Melanin Pigments
At present such great amount of medicinal preparations exists, as never before. As a rule, after some time papers appear in the literature stating an increased sensibility to a new preparation. There were many measures accepted and experiments performed to establish any dependence between the physico-chemical properties and immunogenic activity [Vladimirov V. G., Krasilmikov I. J., Arapov O. B.: Radioprotektory, Naukova dumka, Liev 1980, p. 264; VIDAL cat.xe2x80x94Lekarstvennyie preparaty v Rossii, Astra-Pharm-Servis, Moscow 1997, p. 1166] of medicinal compounds for the purpose of foreseeing and judging their allergicity. To date it is not possible to perform such corrections because of the level of present scientific knowledge. Nevertheless, several facts have been established which may be presented as follows. Immunogenic activity of an antigen depends on its physico-chemical properties and on the ability of the immunising (immunised) organism to response to a given antigen. According to the ability to elicit an immune response the antigens may be classified into two groupsxe2x80x94weak and strong. Among the substances with established chemical nature the strongest immunogens are proteins, although also polysaccharides, synthetic polypeptides and other polymers may become immunogens under certain conditions [Koen S., Word P., Mat-Classen R.: Immunology, Medicina, Moscow 1983, p. 400; Allergeny i immunopatologyia v klinike i experimente, Sbornik nautschnykh trudov, Moscow 1988, p. 164; Buc M. et al.: Klinickxc3xa1 imunolxc3x3gia, Veda, Bratislava 1997, p. 364]. Sufficiently high molecular weight is also a condition for sufficient immunogenecity of antigens. For example, if the molecular weight is less than 10 000, as a rule the substance is weakly immunogenic.
Most of the high-molecular proteins have the molecular weight over 100 000. With decreasing dimensions and molecular weight of antigens individuality of their structure is getting lost, heterogeneity and immunogenic activity decrease. It has been observed that the more complicated is the structure of the molecule of an antigen the more immunogenic it is. An example has been shown in the case of immunogenecity of synthetic polypeptides. If the polypeptide was formed of residues of one amino acid, it was weakly immunogenic. If it consisted of more kinds of residues of two or three amino acids, it gained immunogenic properties. The presence of aromatic amino acids (for example tyrosine) in synthetic polypeptides ensures immunogenicity of the molecule. It has been shown that the ability to elicit creation of antibodies at high levels belongs to substances which have groups, charged at their surface.
Some theories connect the immune activity of compounds also with the strength of their molecule. From this point of view immunogenicity, the ability to induce the cell or humoral immune response, depends on individuality and physico-chemical properties of the antigen, on dimensions of its molecule, on the character, amount and localisation of antigenic determinants in the antigen molecule.
Based on the above given facts and other general knowledge on the properties of animal, microbial, vegetable and synthetic melanins all these substances have the property of immunogenic activity, and presumably they may be classified into the group of weak antigens.
Pharmacological Properties of Melanins
When using melanins for pharmacological reasons, their solubility is of great importance. In the studies, described up to now, melanin preparations have been used in the form of aqueous suspensions or suspensions in physiological saline, they have been applied intramuscularly, but they were practically never absorbed and, therefore, they have had only local effects. The most effective are soluble melanins which are applied perorally or intravenously. Similar preparations have been produced, but they consisted mostly of pseudoglobulin melanoproteins, i.e. complexes of chromogenic parts with proteins which are not able to transport electrons and have a reduced ability to protect against the influence of radiation and toxic free radicals [Buc M. et al.: Klinickxc3xa1 imunolxc3x3gia, Veda, Bratislava 1997, p. 364; Feren{haeck over (c)}ik M., {haeck over (S)}tvrtinovxc3xa1 V., Bernadi{haeck over (c)} M., Jakubovský J., Hulin I.: Zxc3xa1pal, horxc3xa{haeck over (c)}ka, bolest"", Slovak Academic Press, Bratislava 1997, p. 216; Mayer V., Hallauer J., Baum M. K.: Ochorenie, spxc3x4sobenxc3xa9 nxc3xa1ikazou virusom HIV/AIDS, Vydavatel""stvo Slovenskej akadxc3xa9mie vied, Bratislava 1996, p. 364].
There is concern in the investigation of pharmacological activity of water-soluble (chromogenic) part of the pigment, extracted from vegetable culturesxe2x80x94fytomelanin.
Melanin Preparations in the World
At present, especially in the period of the last 15 years, more and more attention is concentrated on melanins as perspective preparations for many areas in industry and medicine. Melanins are produced at present by a number of companies; the best known are the products, produced and distributed by SIGMA CHEMICAL Corp., USA [Sigma Chemical Co.: Biochemikalien und Reagenzien fxc3xcr die naturwissenschaftliche Forschung, Germany 1997, p. 2736; The Merck Index, An encyclopedia of chemicals, drugs and biologicals, 12th Edition, Whitehouse Station, N.Y. 1996, p. 2668].
Up to now production of synthetic melanin (so called DOPA-melanin) and sepiomelanin, isolated from the ink of Sepia officinalis, has been mastered. Nevertheless, because of exotic raw materials the preparations are very expensive, they are produced in insufficient amounts, and they are in general not available for broad usage. Small amounts of several kinds of melanins of animal and microbial origin are produced in laboratories, but because of high complexity of the production and because of lack of basic raw materials they do not represent serious and cheap possibilities of a large scale industrial production. Production of melanins of vegetable origin in chemically pure form is not known up to the present.
All known processes are based on the isolation of melanin by complicated method from biological structures in which they occur or on synthetic methods, i.e. on autooxidation of tyrosine.
Disclosure of the Invention
The above mentioned disadvantages are eliminated to a great extent by biologically active fraction of melanin according to this invention, the structure of which results from polymerisation of vegetable flavonoids, especially of catechins and lecoanto-cyanidines (III). 
As the fraction of melanin is an amorphous substance, it is impossible to determine its precise structure. As an example we present one of the most probable structures (IV), where the arrows indicate further paths of possible polymerization. 
where R is independently OH, Hxe2x95x90O, xe2x80x94COOH or xe2x80x94NH2.
The base for the fytomelanin molecule is a structure, consisting of monomeric units of vegetable flavonoids according to this invention, especially of catechins and lecoantocyanidines. As the polymerization itself and, therefore, the number of possibilities of forming different structures is rather high, the limiting factor here is the molecular weight Mh=(5xc2x11).103 Da.
The empirical formula for one of the most probable structures which is represented by the formula IV is
[C34-59O14-23H32-44N6-8]n, where n=6 to 8
In the next Table 1 there are given physico-chemical properties of the substance, arising as a result of polymerization of vegetable flavonoids according to the technology which we have proposed.
xe2x80x9cMelanins of vegetable originxe2x80x9d is just an overall summary name for dark brown and black pigments of plants which are, from the chemical point of view, products of oxidation of flavan-3,4-diols. Taking into account that the reaction of oxidation polymerization is influenced by a number of factors, such as temperature, pH of the environment, hydromodule, phase ratio, reaction time, content and concentration of components, and the like, also its products may partially differ and they have no strict physico-chemical characteristics or they also differ considerably one from the other.
The structure of melanins is the most probable structure if conditions for polymerization in the course of their synthesis from natural vegetable raw materials are provided as further described in detail, and if corresponding technological procedure is used. These conditions practically eliminate the way of condensation of flavonoids with opening the pyran cycle (the scheme after K. Freidenberg), but they correspond to the reaction according to the scheme after D. E. Katueno with joining molecules xe2x80x9chead to headxe2x80x9d and xe2x80x9ctail to tailxe2x80x9d [Kretovich V. A.: Osnovy biokhimii rastenyi, Vysschayia schkola 1970, p. 540].
Besides, the base of the molecule of known melanins is pyrocatechinol structure (formula II) which arises as a result of the opening of the pyran cycle of the lekoantocyanidine molecules and by the following polymerization of the formed fragments. It can be explained by xe2x80x9charderxe2x80x9d conditions, where one of the main reasons is the usage of high concentration of bases, 0.25 to 1.20 M, of nonadequate thermal modes, usage of non-demineralised water in the whole process or only partially. Moreover, the technology which we have proposed ensures that a structure of a polymer arises having not very high condensation degree (n=5 to 7), in consequence of what it is possible to synthesise chemically homogeneous productxe2x80x94a polymer, fytomelanin having certain physico-chemical and biological characteristics and reproducible properties.
Isolation of natural polymer from biological subjects is practically impossible. This refers especially to melanins of vegetable origin for which there exists no inert solvent. The only solvents are 0.5 to 1.0 M aqueous solutions of NaOH or KOH. Nevertheless, the process of dissolution is not a physical process, but it is actually chemical reaction of a base with a polymer which causes destruction of the native biopolymer. This action severely disturbs the chemical nature, actually leading to decomposition to individual fragments which are further xe2x80x9csewn togetherxe2x80x9d according to one of the known schemes of oxidation polymerization. By the subsequent procedure arising admixtures and other reaction products are eliminated.
The product prepared in this way is no more the original native form, but a synthetic polymer which is also characterised by the properties, belonging to the original melanins and to this class of compounds. In this respect we may, therefore, speak about a synthesis of the product in a biochemical way on the base of the original vegetable raw materials.
Identification
One of the tests for fytomelanin identification are the VIS-spectra. Bright spectral absorption bands:
in the wavelength range of 1600 to 1700 cmxe2x88x921 
in the wavelength range of 3300 to 3500 cmxe2x88x921.
The spectral bands correspond to carbonyl groups (absorption in the range of 1700 cmxe2x88x921), carbon-carbon bonds (absorption in the range of 1600 cmxe2x88x921), NH4 and OHxe2x80x94 groups (absorption in the range of 3300 to 3500 cmxe2x88x921) (Table 1).
The further identification features or characteristics are as follows:
a/ insolubility in water and in most organic solvents
b/ complete solubility in 0.5 M NaOH or KOH,
c/ precipitation from solutions in the presence of FeCl3,
d/ discoloration if strong oxidation agents (KMnO4, H2O2) are used,
e/ ability to regenerate ammonia solution of AgNO3.
The most important characteristics of the fytomelanin is the presence of paramagnetic centres (unpaired free electrons) with a concentration of 1018 to 1022 spin/g.
The Process for production of a fraction of vegetable melanin consists in that the vegetable raw material is treated with a 0.05 to 0.3 M aqueous solution of an alkali metal hydroxide at a temperature of 15 to 75xc2x0 C., pH of the extract is adjusted to 1 to 2 by adding an inorganic acid, based on chlorine, and the excluded sediment is purified and subsequently dried at 100 to 110xc2x0 C.
The purification is preferably performed by washing with a solution of an acid, based on chlorine, having pH of 1.0 to 3, until colourless liquid is reached over the sediment, by subsequent washing with ethanol and with further organic polar solvents. In all of the processes water of pharmacological quality is used.
The dry product may be further subjected to further purification, and semiquinonic radical is activated.
The Process for preparation of a substance the physico-chemical parameters of which correspond to Table 1, consists in the following steps:
The obtained summary product represents a fraction of vegetable melanins, having a molecular weight Mh=(5xc2x12).103 Da. A further stage of the technological process is removing of the residual accompanying admixtures and products of the native polymer degradation.
The final product may be prepared in the following forms:
a) dry amorphous powder with dark brown colour, showing characteristic metallic lustre, insoluble in water
b) dry amorphous powder with dark brown colour, showing characteristic metallic lustre, soluble in water
c) aqueous solution of fytomelanin with maximum concentration up to 3 to 5% by weight
d) paste with dark brown colour, containing 10 to 15% by weight of the basic product.
The nature of the invention consists also in a pharmaceutical preparation which contains a fraction of the vegetable melanin according to this invention and a pharmaceutically acceptable carrier.
It was found that a fraction of the vegetable melanin according to this invention is suitable to be used as a drug, especially as an antioxidant, to block the peroxidation of lipids, to activate leukocytes, to regulate the behaviour of the complement system. Based on the results achieved which are described below and on the description of biological functions of substances having melanin character [Feren{haeck over (c)}ik M., {haeck over (S)}tvrtinovxc3xa1 V., Bernadi{haeck over (c)} M., Jakubovský J., Hulxc3xadn I.: Zxc3xa1pal, horxc3xa{haeck over (c)}ka, bolest"", Slovak Academic Press, Bratislava 1997 ,p 216; Mayer V., Hallauer J., Baum M. K.: Ochorenie, spxc3x4sobenxc3xa9 nxc3xa1kazou vxc3xadrusom HIV/AIDS, Vydavatel""stvo Slovenskej akadxc3xa9mie vied, Bratislava 1996, p. 364; Sigma Chemical Co.: Biochemikalien und Reagenzien fxc3xcr die naturwissenschaftliche Forschung, Germany 1997, p. 2736; {haeck over (D)}ura{haeck over (c)}kovxc3xa1 Z., Bergendi L""., Liptxc3xa1kovxc3xa1 A., Muchovxc3xa1 J.: Free radicals derived from oxygen and medicine, Bratislava Medical Journal 1993, Nr. 8, 419-434; {haeck over (D)}ura{haeck over (c)}kovxc3xa1 Z., Felix K., Fenikovxc3xa1 L"", Kep{haeck over (s)}tovxc3xa1 I., Labuda J., West U.: Superoxide dismutase mimetic activity of a cyclic tetrameric Schiff base N-coordinated Cu(II) complex, BioMetals, Nr. 5, 183-187, 1995; Novxc3xa1k M.: Neuroimunology of the Alzheimer""s Disease, Bratislava Medical journal 1997, 98, 303-314] we can conclude the following:
A damage in a human organism arises through the action of external or inner factors. It should be understood that the term xe2x80x9cdamagexe2x80x9d includes measurable changes at which homeostasis of the intracellular environment is damaged to such a degree that the intracellular structures and the cells themselves are not able to hold and compensate the disorder by their own mechanisms. At this moment a disorder of subcellular structures and loss of cell integrity encounters. If the process affects a sufficiently high number of cells the irreversible cell conditions manifest themselves by the loss of the corresponding organ function. It results in a disorder of organs with subsequent changes of functions of the whole organism.
Melanins, including fytomelanin, belong to a group of substances which take part in the corrective measures. It means that it is not a substance which compensates the loss or disorder in any way. Neither is it a substance which would act as a factor of deficiency supplementation. It is a substance which actively affects the processes which start when homeostasis of cells and subsequently of the whole organs is damaged. In accordance with modern concepts of medicine the solution of the problem of cell and organ damages is a principal solution. This approach observes the modern trends in the development of scientific research in the field of biological-medical sciences. The principle of cell homeostasis loss is the basis for all disorders in the human organism except for genetically conditioned disorders. Also the action of physical factors such as radiation, action of extreme temperatures and physical factors causes disorder of the cell structure. The organism must also in these cases ensure optimisation of conditions in the sense of organism survival. That what happens in the organism subsequently, is a process in which many mechanisms and substances take part. The substance fytomelanin plays crucial role in the process. It follows from the fact that in the damaging process mechanisms are activated which are directed to elimination of the damaging injurer. At the same time substances arise which have high bactericide activity and the ability to react very rapidly with other substances which occur in the place of damage. The substances directed to elimination of the injurer have no ability to recognise the damage of useful and useless parts of the organism. As a final consequence these substances damage all structures occurring at the place of their rise, forming and action. In most cases these are substances with small molecular weight. A similar process of damaging takes place also if a disorder encounters of an organ or tissue which has already been damaged and adapted. For example in the case of atherosclerosis a certain kind of equilibrium state arises between a disordered vessel, blood flow and the tissue which is supplied with blood from the vessel. If the cell integrity is disturbed (preatherosclerotic process), again mechanisms directed to remedy and organism survival start to act in this dramatic situation. These and the above mentioned processes of damage remedy take place with forming reactive oxygen intermediates, superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen and reactive nitrogen intermediates. Based on the present analyses, fytomelanin works as a means for the xe2x80x9ccontrolxe2x80x9d of forming these substances. These substances are able to react with the injurer, but their important role consists in that they trigger a cascade of changes of forming other substances which act as damaging substances and as substances which ensure activation and regulation of the remedy processes. Exchange of intercell information is influenced by these substances. The substances, used for treatment so far, have a character of substances with attenuating or stimulating effect. Other substances have a function of supplementation or competitive inhibition.
Fytomelanin is a substance the effect of which depends on the activation state in the case of damage and on the activation state of the systems taking part in these complex processes (complement system, coagulation system and kinin system). Fytomelanin works really as a regulator which might serve for the complete control of processes which take place spontaneously. For the preparation of a research concept concerning an overall utilisation in medicine directed to elucidation of the role of this molecular fraction of melanins of vegetable origin it is further necessary to perform observations of a model situation on an integral organism (reperfusion damages, oxygen and calcium paradox). These would be studies which should define the participation of fytomelanin in damage and remedy of an organism. Fytomelaninxe2x80x94substance may by especially perspective, its binding to enzymes and to substances, containing metal elements, may result in very broad utilisation. Moreover, a substance which behaves as a process regulator might be useful if used in many lethal diseases and in the elucidation of the origin of many diseases which are not yet explained.
The fytomelanin fraction according to the present invention, as an active substance to be used as a drug, may be used in the following pharmacological forms: water-soluble dry form, injection form, dry substance for peroral administration, solution for injection application, solution for peroral administration, tablets, granules, capsules, dragxc3xa9e, suspensions, syrup, gel, jelly, ointment, creams, solution for external application, solution for infusions, aerosol forms, cosmetic additive for liquids, creams, shampoos.